In the past, pluripotent stem cells have been generated by means of nuclear transplant and cell fusion (Shinya Yamanaka, Pluripotency and Nuclear Reprogramming, Philos Trans R Soc Lond B Biol Sci. 363(1500): 2079-2087 (Jun. 27, 2008)). Both methods require embryonic stem cells, which pose ethical dilemmas for both research and therapeutic use. This issue is overcome by the recently discovered induced pluripotent stem (iPS) cells, which share the same attractive biological properties of embryonic stem (ES) cells (Yamanaka, A Fresh Look at iPS Cells. Cell 137:13-17 (S. 2009)).
Induced pluripotent stem cells were first produced from mouse fibroblasts in 2006 (Takahashi, Y. and S. Yamanaka, Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors, Cell 126: 663-676 (2006)) and human fibroblasts in 2007 (Yu Junying, et al., Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells, Science 318: 1917-1920 (2007), Takahashi, K., et al. Induction of Pluripotent Stem Cells From Adult Human Fibroblasts by Defined Factors, Cell 131: 861-872 (2007)) by forcing the expression of defined factors. Two sets of factors have been used to trigger the reprogramming of adult somatic cells to iPS cells in these studies: one includes Oct-3/4, Sox2, Klf4, and c-Myc (Takahashi, Cell 126: 663-676; Takahashi, Cell 131:861-872); and the other includes Oct4, Sox2, Nanog, and Lin28 (Junying, Cell 318:1917-1920). The reprogramming efficiency of these defined factors can be increased 10-fold by knocking down p53 activity.
In 2008, Melton's group demonstrated that by delivering a specific combination of three transcription factors, Ngn3 (also known as Neurog3), Pdx1, and Mafa, the differentiated pancreatic exocrine cells in adult mice were re-programmed into cells that closely resemble β-cells (Zhou, Q. et al., In Vivo Reprogramming of Adult Pancreatic Exocrine Cells to β-cells, Nature 455: 627-633 (2008)). The induced β-cells are morphologically indistinguishable from endogenous islet β-cells; they express genes essential for β-cell function and can ameliorate hyperglycemia by remodeling local vasculature and secreting insulin. This study suggests that adult somatic cells can be re-programmed to tissue specific cells directly by a specific combination of transcription factors without reversion to a pluripotent stem cell state.
The potential of iPS cells is enormous. However, the clinical application of iPS cells faces many obstacles (Yamanaka, Cell 137: 13-17; Miura, K. et al., Variation in the Safety of Induced Pluripotent Stem Cell Lines, Nature Biotechnology 27(8):743-745 (2009); Carpenter, M. et al., Developing Safe Therapies From Human Pluripotent Stem Cells, Nature Biotechnology 27: 606-613 (2009)). One major hurdle is directly related to the delivery vehicle for the re-programming factors. Initially, the re-programming factor genes were introduced into somatic fibroblast by retro or lentiviral vectors (Takahashi, Cell 126: 663-676; Junying, Cell 318:1917-1920; Takahashi, Cell 131:861-8723-5). The process of re-programming through retro or lentiviral vectors had an efficiency of only ˜0.05% (Okita, K. et al., Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors, Science 322: 949-953; Yamanaka, S. Elite and Stochastic Models for Induced Pluripotent Stem Cell Generation, Nature 460: 49-52 (2009)).
Insertional activation of an oncogene is always a great concern of using retro or lentiviral vectors since these vectors randomly integrate into the host's genome. Although transgenes are largely silenced in iPS cells, reactivation of c-myc transgene could lead to tumorigenesis (Okita, K., et al., Generation of Germline-Competent Induced Pluripotent Stem Cells, Nature 448: 313-318 (2007)). Leaky expression of these transgenes may also inhibit complete iPS cell differentiation and maturation, leading to a greater risk of teratoma formation (Yamanaka, Cell 137:13-17). In addition, the transgenes could be re-activated and expressed in cells that are re-differentiated from the iPS cells, leading to a risk of re-programming differentiation status of iPS cells or tumor formation.
A non-integrating viral vector such as an adenoviral vector was later used to deliver these re-programming factor genes (Zhou, Nature 455: 627-633; Stadtfeld, M. et al., Induced Pluripotent Stem Cells Generated Without Viral Integration, Science 322: 945-949 (2008)). However, large amounts of adenoviral vectors, which may cause cytopathic effect on cells, are required for effective transduction into cell types that lack the adenovirus receptor CAR. In addition, low level expression of some adenoviral genes may affect the transduced cells if a “non-gutless” adenoviral vector is chosen as a delivery vehicle.
Re-programming can also be accomplished via direct plasmid transfection (Okita, Science 322: 949-953; Yu, J. et al. Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences, Science 324: 797-801 (2009)), but it is more than 100-fold less efficient than that of a retroviral vector (Okita, Science 322: 949-953). Both adenoviral vector transduction and plasmid transfection may not exclude stable integration. The integration frequencies of adenoviral vectors are ˜10−3 to 10−5 per cell (Harui, A. et al., Frequency and Stability of Chromosomal Integration of Adenovirus Vectors, J. Virology 73: 6141-6146 (1999)).
A comparison of methods to generate pluripotent stem cells is shown in table 1.
TABLE 1Pros and Cons of three methods to create pluripotent stem cellsUse ofhumanApplication toImmuno-Chromosomeembryohuman cellsrejectionabnormalityNuclear transferYesunknownYesnormalES cell fusionYesYesYestetraploidiPS CellsNoYesNovector insertion(oncogenederegulation)ES, embryonic stem cell;iPS, induced pluripotent stem cells.In 1998, Frankel and Green independently observed that HIV-1 Tat protein can penetrate cells in a receptor-independent fashion (Frankel, A. and C. Pabo, Cellular Uptake of the Tat Protein from Human Immunodeficiency Virus, Cell 55: 1189-1193 (1988); Green, M. and P. Loewenstein, Autonomous Functional Domains of Chemically Synthesized Human Immunodeficiency Virus Tat Trans-Activator Protein, Cell 55:1179-1188 (1988)). The Tat protein transduction domain (PTD) that contains the short basic arginine-rich region (aa 48-57) of HIV-1 Tat is widely used to deliver variety of molecules including peptides both in vitro and in vivo (Schwarze, S. R. et al., In Vivo Protein Transduction: Delivery of a Biologically Active Protein Into Mouse, Science 285: 1569-1572 (1999); Lindsay, M. A. Peptide-Mediated Cell Delivery: Application in Protein Target Validation, Current Opinion in Pharmacology 2:587-594 (2002); Kwon, Y. D., et al., Cellular Manipulation of Human Embryonic Stem Cells by Tat-Pdx1 Protein Transduction, Molecular Therapy 12: 28-32 (2005); Kim, D., Generation of Human Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins, Cell Stem Cell 4: 472-476 (2009); Wadia, J. S. et al., Transducible Tat-HA Fusogenic Peptide Enhances Escape of Tat-Fusion Protein After Lipid Raft Macropinocytosis, Nature Medicine 10:310-315 (2009); Zhou, H. et al., Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins, Cell Stem Cell 4: 381-384.19-24 (2009)). This method to deliver molecules by a protein-based vehicle is called protein transduction. Binding of Tat-PTD to cell surface through ionic interaction leads to the internalization of Tat-fusion proteins by lipid raft-dependent macropinocytosis (Wadia, J. S., Nature Medicine 10:310-315). The majority of the Tat-fusion protein, however, remains trapped in macropinosomes, indicating that the escape of peptides or proteins from macropinosomes is an inefficient process (Wadia, J. S., Nature Medicine 10:310-315). Recently, poly-arginine (11R or 9R) PTD fused to C-terminus of the 4 re-programming factors (Oct4, Sox2, Klf4, and C-Myc) successfully re-programmed mouse embryonic fibroblast (Zhou, H. et al., Generation of Induced Pluripotent Stem Cells using Recombinant Proteins, Cell Stem Cell 4: 381-384 (2009)) and human newborn fibroblast (Kim, D., Cell Stem Cell 4: 472-476) cells to iPS cells but with very low efficiency.